The CNS actions of cannabis and related compounds (cannabinoids) are thought to be primarily mediated by the CB1 subtype of cannabinoid receptor. However, an increasing number of functional studies have identified novel cannabinoid-like actions in the brain, which point to the involvement of additional receptors. Recently it has been suggested that several cannabinoid receptor ligands bind to the orphan G-protein coupled receptor GPR55. As GPR55 mRNA is expressed within human brain tissue, this receptor may therefore play a role in mediating some of the CNS actions of cannabinoids. In this study we have assessed a range of cannabinoid ligands in their ability to provoke agonist-induced trafficking and the cellular localization of recombinant GFP, HA and SEP (pH sensitive GFP) fusions of GPR55. The constructs were transfected into neurons (5-14 days in vitro) or cell lines (HEK293 and COS7). In cell lines, treatment with cannabinoid ligands results in a rapid redistribution of HA and GFP fusions into endosomes, however the pharmacology of GPR55 trafficking differs from that for the classical CB1 receptor. When expressed in neurons GPR55-GFP is associated with the somatodendritic plasma membrane and using an antibody to GFP we find that GPR55 is expressed at the cell surface on both axons and soma/dendrites, whereas CB1 is polarized to the axonal surface. This profile was further established following expression of SEP-GPR55 or SEP-CB1, which reveals receptor expression on the cell surface of live neurons. In conclusion, both CB1 and GPR55 internalize following prolonged agonist exposure, which may contribute to cannabinoid tolerance in vivo. The differing cellular distributions of CB1 and GPR55, suggests that they sub-serve distinct functional roles in the CNS, with the potential for both receptors to act presynaptically, but with GPR55 mediating postsynaptic actions of cannabinoids.